CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling

Abstract

Cancer stem cells (CSCs) are characterized by their self-renewal and differentiation abilities. CD44v6 is a novel CSC marker that can activate various signaling pathways. Here, we hypothesized that the HGF/Met signaling pathway promotes stemness properties in CD44v6+ hepatocellular carcinoma (HCC) cells via overexpression of the transcription factor, cJun, thus representing a valuable target for HCC therapy. Magnetic activated cell sorting was used to separate the CD44v6+ from CD44v6- cells, and Met levels were regulated using lentiviral particles and the selective Met inhibitor, PHA665752. An orthotopic liver xenograft tumor model was used to assess the self-renewal ability of CD44v6+ cells in immunodeficient NOD/SCID mice. Luciferase reporter and chromatin immunoprecipitation assays were also conducted using cJun-overexpressing 293 T cells to identify the exact binding site of cJun in the Nanog promoter. Our data demonstrate that CD44v6 is an ideal surface marker of liver CSCs. CD44v6+ HCC cells express higher levels of Met and possess self-renewal and tumor growth abilities. Xenograft liver tumors were smaller in nude mice injected with shMet HCC cells. Immunohistochemical analysis of liver tissue specimens revealed that high Met levels in HCC cells were associated with poor patient prognosis. Further, a cJun binding site was identified 1700 bp upstream of the Nanog transcription start site and mutation of the cJun binding site reduced Nanog expression. In conclusion, the HGF/Met signaling pathway is important for maintenance of stemness in CD44v6+ HCC cells by enhancing expression of cJun, which binds 1700 bp upstream of the Nanog transcription start site.

Exosome-derived tRNA fragments tRF-GluCTC-0005 promotes pancreatic cancer liver metastasis by activating hepatic stellate cells

“…Lentivirus-mediated tRF-GluCTC-0005 expression or silencing was achieved by the documented method [ 34 ] with some modifications. Briefly, for expressing, the synthesized tRF-GluCTC sequence was inserted into pLKD-CMV-mcherry-2A-Neo-U6-shRNA vector containing the miR-30 backbone (ObiO Technology).…”

“…Total RNA extracted from cell lines and pancreatic tissue specimens with Trizol reagent (Invitrogen) was reverse transcribed with random primers or specific tRF stem-loop RT primers. Real-time quantitative PCR using SYBR Premix (DRR041A, TaKaRa, Japan) was performed as described previously [ 34 ]. U6 RNA was used as an internal control for the quantification of tRFs.…”

“…The cell invasive and migratory ability was detected using an 8um pored, 6.5 mm polycarbonate transwell filter (Millipore, Billerica, MA, USA), according to our previous study [ 34 ]. For the migration assay, 5×10 4 cells were seeded in the upper chamber with serum-free DMEM.…”

“…Western blot analysis was performed as described previously [ 34 ]. Primary antibodies are listed in the Supplementary Table S2 .…”

Cancer stem cells: a target for overcoming therapeutic resistance and relapse

“…CD44v8-10 have been identified as human gastric CSC markers that contribute to tumor initiation 306 . CD44v6+ has also been reported as a CSC marker for colon cancer and HCC 90 , 307 . CD44s, CD44v4, and CD44v9 at the invasive tumor front are associated with poor prognosis in gastric cancer patients and CD44s-expressing CSCs exhibit mesenchymal properties 308 .…”